Methods and Compositions for the Selection of a Transgenic Plant

Abstract

Compositions and methods are provided to screen, identify, select, isolate, and/or regenerate targeted integration events using seed priming. Seed priming provides the identification of a seed having stably incorporated into its genome a site-specific recombinase mediated integration of a selectable marker at a target locus operably linked to a promoter active in the seed.

Description

Claims

1. A method to identify a plant having a DNA construct stably incorporated in its genome comprising: (a) providing a population of seeds, wherein at least one seed in the population has stably incorporated into its genome the DNA construct comprising the following operably linked components in the following order: a promoter active in the seed, a first recombination site, a polynucleotide encoding a first selection marker that confers resistance to a selective agent, and a second recombination site;(b) contacting the population of seeds with a priming matrix comprising an effective concentration of the selection agent for a time sufficient to produce a population of primed seeds; and,(c) incubating the population of primed seed under germination conditions, thereby identifying plants having the DNA construct stably incorporated into their genome.

2. The method of claim 1, wherein the first and the second recombination sites are dissimilar and non-recombinogenic with each other.

3. The method of claim 1, wherein the selective agent comprises an herbicide, a growth regulator, or an antibiotic.

4. The method of claim 3, wherein the selective agent is selected from the group consisting of glufosinate, phosphinothricin, glyphosate, bromoxynil, methotrexate, imidazolinones, sulfonylureas, cyanamide, kanamycin, G418, neomycin, and hygromycin B.

5. The method of claim 3, wherein the first selection marker is selected from the group consisting of Bar, phosphinothricin acetyltransferase (PAT), glyphosate oxidoreductase (GOX), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), CP4, glyphosate N-acetyltransferase (GAT), bromoxynil nitrilase (BXN), dihydrofolate reductase, acetolactate synthase (ALS), cyanamide hydratase (CAH), neomycin phosphotransferase (nptil), aminoglycoside 3′-phosphotransferase (APH3′ II), and hygromycin phosphotransferase (hph).

6. The method of claim 5, wherein the polynucleotide encoding the first selection marker has been synthesized using maize preferred codons.

7. The method of claim 1, wherein the DNA construct comprises in the following order: a first expression unit comprising the promoter active in the seed operably linked to the first recombination site operably linked to the polynucleotide encoding the first selection marker; and a second expression unit comprising in the following order, a second promoter active in the plant operably linked to a polynucleotide of interest, and the second recombination site.

8. The method of claim 7, wherein the polynucleotide of interest encodes a second selection marker, wherein the second selection marker is distinct from the first selection marker by having a different selection means.

9. The method of claim 1, wherein the plant further has stably incorporated into its genome a polynucleotide encoding a site specific recombinase.

10. The method of claim 9, wherein the site specific recombinase comprises a FLP recombinase, a Cre recombinase, a lambda integrase, a SSV1 integrase, or a phiC31 integrase.

11. The method of claim 1, wherein at least one of the first or the second recombination sites are selected from the group consisting of a lox site, a mutant lox site, a FRT site, a mutant FRT site, an att site, and a mutant att site.

12. The method of claim 1, wherein the plant is maize, wheat, rice, sorghum, rye, oat, barley, millet, Brassica, alfalfa, sunflower, safflower, soybean, tobacco, cotton, or Arabidopsis.

13. A method of priming a seed comprising (a) providing a seed having stably incorporated into its genome a DNA construct comprising, in the following order, a promoter active in the seed operably linked to a first recombination site operably linked to a polynucleotide encoding a selection marker that confers resistance to a selective agent; and(b) contacting the seed with a priming matrix comprising an effective concentration of the selective agent for a time sufficient to produce a primed seed.

14. The method of claim 13, wherein the DNA construct comprises in the following order the promoter operably linked to the first recombination site operably linked the polynucleotide encoding the selection marker, and a second recombination site.

15. The method of claim 14, wherein the first and the second recombination sites are dissimilar and non-recombinogenic with each other.

16. A composition comprising a seed having stably incorporated into its genome a DNA construct comprising, in the following order, a promoter active in the seed operably linked to a first recombination site operably linked to a polynucleotide encoding a selection marker that confers resistance to a selective agent; and, a priming matrix comprising an effective concentration of the selective agent.

17. The composition of claim 16, wherein the DNA construct comprises in the following order, the promoter operably linked to the first recombination site operably linked to the polynucleotide encoding the selection marker, and a second recombination site.

18. The composition of claim 17, wherein the first and the second recombination sites are dissimilar and non-recombinogenic with each other.

19. The composition claim 16, wherein the DNA construct further comprises a polynucleotide of interest.

20. The composition of any one of claim 16, wherein the DNA construct further comprises a second selection marker.

21. The composition of claim 20, wherein the second selection marker confers resistance to a second selective agent.

22. The composition of claim 20, wherein the priming matrix further comprises the second selective agent.

23. The composition of claim 16, wherein the seed is from a plant selected from the group consisting of maize, wheat, rice, sorghum, rye, oat, barley, millet, Brassica, alfalfa, sunflower, safflower, soybean, tobacco, cotton, and Arabidopsis.